Interferon (IFN) is an antiviral cytokine that mediates its activity through the induction of 300+ interferon-stimulated genes (ISGs). Several of these genes are well-known inhibitors of virus replication, but the majority of the ISGs remain uncharacterized with respect to their antiviral potential. Microarray and knock-out studies suggest that many of these ISGs possess significant antiviral functions. The long-term objectives of this project are to identify and characterize ISGs that exhibit novel antiviral activity in response to hepatitis C virus (HCV), the etiological agent of non-A, non-B viral hepatitis. Our experimental approach relies on generating a collection of lentiviral-based vectors that express all known ISGs. The collection will be screened in a high-throughput assay for genes that inhibit HCV replication when overexpressed. We will take advantage of the recently developed cell culture model of HCV infection to carry out this screen. Candidate anti-HCV ISGs will be further characterized with functional assays to determine their mechanisms of action. We also plan to utilize our screen-based approach to identify ISGs that exhibit novel antiviral activity against different virus families. Several significant human pathogens, including Dengue virus (DV) and influenza A virus (FluA), will be included in our analysis. Data from representative members of each virus family will be assembled into an "ISG profile," which will be used for cross-family comparative analysis. This research is highlighted by the fact that although viruses are significant contributors to human disease, antiviral therapies such as IFN are often ineffective and toxic. HCV affects approximately 3 percent of the world's population and contributes significantly to cirrhosis and liver cancer;DV is a global health concern that infects millions of people and causes dengue fever and the severe dengue hemorrhagic fever;FluA results in the death of more than 30,000 people each year in the US alone. Identifying genes that exhibit antiviral activity against these and other viruses is important for generating novel, highly effective antiviral therapeutic agents.